Method for producing steviol and steviol glycoside using AOBGL1 homolog

ABSTRACT

The present invention provides a method for producing a steviol glycoside and/or steviol, said method including a step in which a steviol glycoside having at least one unbranched β1,2-glycosidic bond is reacted with the glycosidase AOBGL1 and/or AOBGL3, or a variant thereof, so as to cleave the β1,2-glycosidic bond.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named “P54610_SequenceListing” that is 49.4 KB in size and was created on Feb. 27, 2020, and that was electronically submitted via EFS-Web on Mar. 2, 2020, is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a method for producing a steviol glycoside and steviol.

BACKGROUND ART

The leaves of Stevia rebaudiana of the Asteraceae family contain a secondary metabolite called “steviol” which is a kind of diterpenoid. Steviol glycosides, which are products of the addition of sugars to steviol, include those having sweetness about 300 times higher than that of table sugar. Such steviol glycosides are used as non-caloric sweeteners in the food industry. Obesity is becoming more of a serious social issue on an international scale, and non-caloric sweeteners are increasingly demanded from the viewpoint of promotion of health and reduction of medical cost. Currently, aspartame and acesulfame potassium, which are artificially-synthesized amino acid derivatives, are used as artificial sweeteners. However, naturally-occurring non-caloric sweeteners such as steviol glycosides are expected to be safer and gain more public acceptance.

Among steviol glycosides, stevioside is a compound in which three glucose units are added to steviol, and is contained in the largest amount in the leaves of common Stevia rebaudiana. Stevioside has a degree of sweetness about 300 times higher than that of sucrose, but has slightly bitter taste. Rebaudioside A, which is another steviol glycoside, is a compound in which four glucose units are added to steviol, and has a degree of sweetness about 400 times higher than that of sucrose. Stevioside and rebaudioside A are primary substances responsible for the sweetness of Stevia rebaudiana. There are also known glycosides such as rebaudioside D in which five glucose units are added to steviol and rebaudioside M in which six glucose units are added to steviol. It is also known that Rubus suavissimus contains rubusoside in which one glucose unit is added at each of the 13 and 19 positions of steviol and that this rubusoside is a primary sweet component of Rubus suavissimus. In addition to the above glycosides, glycosides considered to be reaction intermediates and analogs differing in the type of sugar are known to exist (FIG. 1).

Meanwhile, steviol is known to have, for example, improving effect on cognitive function.

If an enzyme acting only on a specific glycoside bond in steviol glycosides can be used, production of a specific glycoside or elimination of an unnecessary glycoside will become possible. This will bring a lot of merits such as facilitating the improvement in taste of Stevia rebaudiana extracts or the purification of a specific steviol glycoside.

An enzyme activity to hydrolyze steviol glycosides has been reported to be observed in some organism species. In particular, concerning the production of steviol glycoside-hydrolyzing enzymes by filamentous fungi of the genus Aspergillus, it has been reported that raw soy sauce has an activity to hydrolyze stevioside into rubusoside (Non Patent Literature 1) and that a pectinase enzyme agent, hesperidinase enzyme agent, and takadiastase enzyme agent have an activity to hydrolyze stevioside into steviol (Non Patent Literatures 2 to 4). A method has also been reported in which steviol is produced from stevioside by the combined use of a pectinase enzyme agent derived from filamentous fungi of the genus Aspergillus and an enzyme agent derived from Helix pomatia (Patent Literature 1). Viscozyme L (novozyme), an enzyme agent derived from Aspergillus aculeatus, has been described to have an activity to hydrolyze stevioside into rubusoside and then into steviol monoglycosyl ester (Non Patent Literature 5). Additionally, an extract obtained from Aspergillus aculeatus by solid culture has been described to have an activity to convert stevioside into steviol (Non Patent Literature 6).

It has been reported that the β-glucosidase of the glycoside hydrolase (GH) family 3 encoded by the AO090009000356 gene of koji mold hydrolyzes disaccharides with a β-glucoside bond (Non Patent Literature 7). Specifically, its specificity for hydrolysis is the highest for laminaribiose with a β-1,3 linkage, followed by β-gentiobiose with a β-1,6 linkage, cellobiose with a β-1,4 linkage, and sophorose with a β-1,2 linkage. However, there has been no report on whether the β-glucosidase has an activity to hydrolyze terpene glycosides typified by steviol glycosides.

Some other organisms have also been reported to have an activity to hydrolyze steviol glycosides. For example, it has been disclosed that bacteria of the genus Clavibacter have an enzyme that decomposes the glucosyl ester bond at the 19 position of rubusoside but does not decompose the glucoside bond at the 13 position (Patent Literature 2). Additionally, it has been reported that Flavobacterium johnsoniae-derived β-glucosidase has an activity to decompose steviol glycosides (an activity to hydrolyze the β-glucoside bond at the 13 position and the glucosyl ester bond at the 19 position).

Although these have been found to have an activity to hydrolyze steviol glycosides, the gene responsible for this activity has not been identified.

Moreover, koji mold contains a large number of genes considered to encode GH3 family or GH5 family enzymes having β-glucosidase-like activity, and thus, even if an enzyme activity can be detected, it is not easy to determine which gene is responsible for the activity.

CITATION LIST Patent Literature

-   Patent Literature 1: National Publication of International Patent     Application No. 2013-516963 -   Patent Literature 2: Japanese Patent Laid-Open No. 10-276775 -   Patent Literature 3: Japanese Patent Laid-Open No. 10-276775

Non Patent Literature

-   Non Patent Literature 1: Journal of the Japanese Society for Food     Science and Technology, vol. 37, No. 5, 369-374 (1990) -   Non Patent Literature 2: Phytochemistry, 6, 1107 (1967) -   Non Patent Literature 3: Journal of the Pharmaceutical Society of     Japan, 95, 1507 (1975) -   Non Patent Literature 4: Journal of the Chemical Society of Japan,     1981, 726 (1981) -   Non Patent Literature 5: J. Agric. Food Chem., 60, 6210-6216 (2012) -   Non Patent Literature 6: Wei Sheng Wu Xue Bao, 54(1), 62-68 (2014) -   Non Patent Literature 7: Biochim Biophys Acta., 1764 972-978 (2006)

SUMMARY OF INVENTION Technical Problem

Under such circumstances, there is a need for a novel method for producing Steviol glycosides and Steviol.

Solution to Problem

The present inventors conducted extensive research to solve the aforementioned problem, and found that a koji mold-derived glycoside hydrolase homolog protein encoded by AOBGL1 gene has an activity to hydrolyze steviol glycosides. That is, the present inventors have found that the protein has an activity to specifically cleave an unbranched β-1,2 bond of a steviol glycoside, thus completing the present invention. Additionally, the present inventors have succeeded in producing a steviol glycoside and/or steviol by further expressing another koji mold-derived glycosidase homolog protein encoded by AOBGL3 gene, thus completing the present invention.

In summary, the present invention is as set forth below.

[1]

A method for producing a steviol glycoside having no unbranched β-1,2-glucoside bond comprising reacting a protein selected from the group consisting of proteins (a) to (c) shown below with a steviol glycoside having at least one unbranched β-1,2-glucoside bond, thereby cleaving said unbranched β-1,2-glucoside bond:

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;

(b) a protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and

(c) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside.

[2]

The method according to [1] above, wherein the protein selected from the group consisting of the proteins (a) to (c) further comprises a secretory signal peptide.

[3]

The method according to [1] above, wherein the secretory signal peptide is a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 28, 30, 32, 34, and 36.

[4]

The method according to [3] above, wherein the steviol glycoside having at least one unbranched β-1,2-glucoside bond is selected from rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside E, stevioside, and steviolbioside.

[5]

The method according to [4] above, wherein the steviol glycoside having no unbranched β-1,2-glucoside bond is selected from rubusoside, rebaudioside A, and steviolmonoside.

[6]

A method for producing a steviol glycoside having no unbranched β-1,2-glucoside bond comprising culturing a non-human transformant obtained by introducing a polynucleotide selected from the group consisting of polynucleotides (a) to (e) shown below into a host producing a steviol glycoside having at least one unbranched β-1,2-glucoside bond:

(a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;

(b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;

(c) a polynucleotide encoding a protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside;

(d) a polynucleotide encoding a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and

(e) a polynucleotide which hybridizes under highly stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and which encodes a protein having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside.

[7]

The method according to [6] above, wherein the polynucleotide selected from the group consisting of the polynucleotides (a) to (e) further comprises a polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide.

[8]

The method according to [7] above, wherein the polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide is a polynucleotide consisting of the nucleotide sequence set forth in any one of SEQ ID NOS: 27, 29, 31, 33, and 35.

[9]

The method according to any one of [6] to [8] above, wherein the polynucleotide is inserted into an expression vector.

[10]

The method according to any one of [6] to [9] above, wherein the transformant is transformed koji mold, transformed yeast, or a transformed plant.

[11]

A method for producing a steviol glycoside having no unbranched β-1,2-glucoside bond comprising contacting an enzyme agent derived from a non-human transformed cell obtained by introducing, into a host cell, a polynucleotide selected from the group consisting of polynucleotides (a) to (e) shown below, with a steviol glycoside having at least one unbranched β-1,2-glucoside bond, thereby cleaving said β-1,2-glucoside bond:

(a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;

(b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;

(c) a polynucleotide encoding a protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside;

(d) a polynucleotide encoding a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and

(e) a polynucleotide which hybridizes under highly stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and which encodes a protein having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside.

[12]

The method according to [11] above, wherein the polynucleotide selected from the group consisting of the polynucleotides (a) to (e) further comprises a polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide.

[13]

The method according to [12] above, wherein the polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide is a polynucleotide consisting of the nucleotide sequence set forth in any one of SEQ ID NOS: 27, 29, 31, 33, and 35.

[14]

The method according to any one of [11] to [13] above, wherein the polynucleotide is inserted into an expression vector.

[15]

The method according to any one of [11] to [14] above, wherein the transformed cell is transformed koji mold, a transformed bacterium, or transformed yeast.

[16]

The method according to any one of [11] to [15] above, wherein the steviol glycoside having at least one unbranched β-1,2-glucoside bond is selected from rebaudioside D, rebaudioside E, stevioside, and steviolbioside.

[17]

The method according to [16] above, wherein the steviol glycoside having no unbranched β-1,2-glucoside bond is selected from rubusoside, rebaudioside A, stevioside, and steviolmonoside.

[18]

A method for producing at least one of rebaudioside B and steviol comprising reacting a protein selected from the group consisting of proteins (a) to (c) shown below, a protein selected from the group consisting of proteins (d) to (f) shown below, and a steviol glycoside having at least one unbranched β-1,2-glucoside bond:

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;

(b) a protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside;

(c) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside;

(d) a protein consisting of the amino acid sequence of SEQ ID NO: 7 or 8;

(e) a protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 7 or 8, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside; and

(f) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 7 or 8, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside.

[19]

The method according to [18] above, wherein the steviol glycoside having at least one unbranched β-1,2-glucoside bond comprises one or more steviol glycosides selected from rebaudioside A, rebaudioside D, stevioside, rubusoside, steviolmonoside, and steviol monoglucosyl ester.

Advantageous Effects of Invention

According to the present invention, there is provided a novel method for producing steviol and a steviol glycoside. With the method according to the present invention, steviol can be produced. Additionally, a steviol glycoside in which a branched trisaccharide (β-1,2 or β-1,3) is added at the 13 position and/or 19 position of steviol can be efficiently extracted and purified.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the structure of various steviol glycosides and the biosynthetic pathway from Stevia rebaudiana.

FIG. 2 shows amino acid sequences and nucleotide sequences of secretory signals, wherein (A) shows MF(ALPHA)1 (YPL187W), (B) shows PHO5 (YBR093C), and (C) shows SUC2 (YIL162W).

DESCRIPTION OF EMBODIMENTS

The present invention will be hereinafter described in detail. The following embodiments are illustrative of the present invention, and are not intended to limit the present invention. The present invention can be carried out in various manners, without departing from the gist of the invention.

Note that all documents, as well as laid-open application publications, patent application publications, and other patent documents cited herein shall be incorporated herein by reference. The present specification incorporates the contents of the specification and the drawings of Japanese Patent Application No. 2015-214255, filed on Oct. 30, 2015, from which the present application claims priority.

“AOBGL1” designates a koji mold-derived β-glucosidase; the cDNA sequence, the genomic DNA sequence, the amino acid sequence, and the amino acid sequence of the mature protein are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

“AOBGL3” designates a koji mold-derived β-glucosidase; the cDNA sequence, the genomic DNA sequence, the amino acid sequence, and the amino acid sequence of the mature protein are shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

1. Method for Producing a Steviol Glycoside

The present invention provides a method for producing a steviol glycoside comprising reacting a protein selected from the group consisting of proteins (a) to (c) shown below (hereinafter referred to as “the protein of the present invention”) with a steviol glycoside having at least one unbranched β-1,2-glucoside bond, thereby cleaving said unbranched β-1,2-glucoside bond.

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;

(b) a protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and

(c) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside.

While the protein shown in (b) or (c) above is typically a variant of a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4, these proteins also include proteins that can be artificially obtained using site-directed mutagenesis as described in, for example, “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 4, Cold Spring Harbor Laboratory Press 2012”, “Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997”, “Nuc. Acids. Res., 10, 6487 (1982)”, “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)”, “Gene, 34, 315 (1985)”, “Nuc. Acids. Res., 13, 4431 (1985)”, and “Proc. Natl. Acad. Sci. USA, 82, 488 (1985)”.

Examples of the “protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside” include a protein consisting of an amino acid sequence wherein, for example, 1 to 77, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1 to 55, 1 to 50, 1 to 49, 1 to 48, 1 to 47, 1 to 46, 1 to 45, 1 to 44, 1 to 43, 1 to 42, 1 to 41, 1 to 40, 1 to 39, 1 to 38, 1 to 37, 1 to 36, 1 to 35, 1 to 34, 1 to 33, 1 to 32, 1 to 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9 (one to several), 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid residue has been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside. In general, the number of deleted, substituted, inserted, and/or added amino acid residues is preferably smaller.

Examples of such proteins include a protein having an amino acid sequence sharing 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity with the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside. In general, the value of sequence identity is preferably greater.

As used herein, the phrase “activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside” refers to the activity to cleave an unbranched β-1,2-glucoside bond formed between two glucose residues in a steviol glycoside (a glycoside wherein glucose is linked to the aglycone, steviol). As used herein, the phrase “unbranched β-1,2-glucoside bond” refers to a β-1,2-glucoside bond wherein the β-1,2-glucoside-bonded glucose residues have no branched structure such as a β-1,3-glucoside bond (“Glc-β1,2-Glu” in Table 1 below). By contrast, when the β-1,2-glucoside-bonded glucose residues further have a branched structure such as a β-1,3-glucoside bond, the β-1,2-glucoside bond can be referred to as a branched β-1,2-glucoside bond (“Glc-β1,2-Glu (β1,3-Glu)” in Table 1 below).

The activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside can be confirmed by reacting the protein of the present invention with a steviol glycoside having at least one unbranched β-1,2-glucoside bond, such as stevioside, purifying the resulting reaction product, and analyzing the purified product using a known technique such as liquid chromatography (LC).

The activity to hydrolyze a β-glucoside bond can be detected by using a medium containing X-β-Glc to culture a transformant genetically modified to express the protein of the present invention and confirming whether the cells and surrounding regions are stained blue. When X-β-Glc is hydrolyzed in the medium, the cells and surrounding regions are stained blue; thus, having blue color is considered to indicate the presence of the activity to hydrolyze a β-glucoside bond.

Alternatively, the activity to hydrolyze a β-glucoside bond can be examined by using p-nitrophenyl β-D-glucopyranoside as a substrate to measure the amount of p-nitrophenol produced by hydrolysis in terms of absorbance (A405).

The phrase “an amino acid sequence wherein 1 to 77 amino acid residues have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4” means that 1 to 77 amino acid residues have been deleted, substituted, inserted, and/or added at any 1 to 77 positions in the same sequence, wherein two or more of deletion, substitution, insertion, and addition may occur simultaneously.

Examples of amino acid residues that are interchangeable are shown below. The amino acid residues included in the same group are interchangeable.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, and cyclohexylalanine;

Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, and 2-aminosuberic acid;

Group C: asparagine and glutamine;

Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, and 2,3-diaminopropionic acid;

Group E: proline, 3-hydroxyproline, and 4-hydroxyproline;

Group F: serine, threonine, and homoserine; and

Group G: phenylalanine and tyrosine.

The protein of the present invention in some embodiments does not contain a secretory signal peptide, because the secretory signal peptide is cleaved. Some other proteins of the present invention may further contain a secretory signal peptide, because the secretory signal peptide remains uncleaved. When the protein of the present invention contains a secretory signal peptide, it preferably contains the secretory signal peptide at its N-terminus. The secretory signal peptide refers to a peptide domain that serves to cause extracellular secretion of a protein bound to the secretory signal peptide. Amino acid sequences of such secretory signal peptides and polynucleotide sequences encoding such amino acid sequences have been well known and reported in the art.

The protein of the present invention can be obtained by, for example, expressing a polynucleotide encoding this protein (see “the polynucleotide of the present invention” described below) in appropriate host cells, although it can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). The protein of the present invention can also be chemically synthesized using a peptide synthesizer from AAPPTec LLC, Perkin Elmer Inc., Protein Technologies Inc., PerSeptive Biosystems, Applied Biosystems, or SHIMADZU CORPORATION, for example.

As used herein, the term “steviol glycoside” refers to a glycoside wherein glucose is linked to the aglycone, steviol. Examples of steviol and steviol glycosides are represented by the following formula (I).

TABLE 1 Compound Name R₁ R₂ Rebaudioside D Glc-β1,2-Glu (β1,3-Glu) Glc-β1,2-Glu Rebaudioside E Glc-β1,2-Glu Glc-β1,2-Glu Rebaudioside A Glc-β1,2-Glu (β1,3-Glu) Glc- Stevioside Glc-β1,2-Glu Glc- Rubusoside Glc- Glc- Steviol H Glc- monoglucosyl ester Rebaudioside B Glc-β1,2-Glu (β1,3-Glu) H Steviolbioside Glc-β1,2-Glu H Steviolmonoside Glc- H Steviol H H

In the table shown above, “Glc” designates glucose, and “Glc-β1,2-Glu” designates the inclusion of a β-1,2-glucoside bond. “Glc-β1,2-Glu (β1,3-Glu)” designates the inclusion of a β-1,2-glucoside bond and β-1,3-glucoside bond.

As used herein, the term “steviol glycoside having at least one unbranched β-1,2-glucoside bond” refers to a steviol glycoside represented by the above general formula (I) wherein at least one of the groups R₁ and R₂ has an unbranched structure. An example thereof is a steviol glycoside selected from rebaudioside D, rebaudioside E, stevioside, and steviolbioside.

The method for producing a steviol glycoside according to the present invention cleaves the unbranched β-1,2-glucoside bond, thereby producing another steviol glycoside.

The steviol glycoside obtained by the present method (hereinafter referred to as “the steviol glycoside of the present invention”) varies depending on the starting material, the “steviol glycoside having at least one unbranched β-1,2-glucoside bond”. Examples thereof are shown below.

TABLE 2 Steviol Glycoside of Starting Material Present Invention Rebaudioside D Rebaudioside A Rebaudioside E Stevioside, Rubusoside Stevioside Rubusoside Steviolbioside Steviolmonoside

In the method for producing a steviol glycoside according to the present invention, the steviol glycoside having at least one unbranched β-1,2-glucoside bond for use as the starting material can be obtained by extraction from Stevia rebaudiana or Rubus suavissimus followed by purification using known methods including extraction with an appropriate solvent (an aqueous solvent such as water, or an organic solvent such as an alcohol, ether, or acetone), a gradient between water and ethyl acetate or other organic solvent, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), and ultra (high) performance liquid chromatography (UPLC). Alternatively, a commercially-available product may be used as the steviol glycoside having at least one unbranched β-1,2-glucoside bond for use as the starting material.

In some embodiments of the present invention, an unbranched β-1,2-glucoside bond of a steviol glycoside selected from stevioside, rebaudioside D, and steviolbioside is cleaved to produce a steviol glycoside selected from rubusoside, rebaudioside A, and steviolmonoside. In another embodiment, an unbranched β-1,2-glucoside bond of rebaudioside E is cleaved to produce rubusoside.

The method for producing a steviol glycoside according to the present invention comprises reacting the protein of the present invention with a steviol glycoside having at least one unbranched β-1,2-glucoside bond, thereby cleaving said unbranched β-1,2-glucoside bond. The method of the present invention may further comprise purifying the steviol glycoside produced in the above step.

The steviol glycoside produced in the above step can be purified using known methods including extraction with an appropriate solvent (an aqueous solvent such as water, or an organic solvent such as an alcohol, ether, or acetone), a gradient between water and ethyl acetate or other organic solvent, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), and ultra (high) performance liquid chromatography (UPLC).

A different steviol glycoside can be produced by combining the protein of the present invention with another protein having a different activity.

Thus, in another embodiment, there is provided a method for producing at least one of rebaudioside B and steviol comprising reacting the protein of the present invention, a protein selected from the group consisting of proteins (d) to (f) shown below (hereinafter referred to as “the second active protein”), and a steviol glycoside having at least one unbranched β-1,2-glucoside bond:

(d) a protein consisting of the amino acid sequence of SEQ ID NO: 7 or 8;

(e) a protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 7 or 8, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside; and

(f) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 7 or 8, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside.

In this method, the steviol glycoside having at least one unbranched β-1,2-glucoside bond may comprise one or more steviol glycosides selected from rebaudioside A, rebaudioside D, stevioside, rubusoside, steviolmonoside, and steviol monoglucosyl ester.

As used herein, the phrase “activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside” refers to a monoglucoside bond and/or monoglucosyl ester bond in a steviol glycoside, which is a glycoside wherein glucose is linked to the aglycone, steviol. In an embodiment, the bond to be cleaved is, but not limited to, a monoglucoside bond at the 13 position. In another embodiment, the bond to be cleaved is, but not limited to, a monoglucosyl ester bond at the 19 position.

The activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside can be confirmed by reacting the second active protein with a steviol glycoside (rubusoside, for example) having either a monoglucoside bond or monoglucosyl ester bond or both of the bonds, purifying the resulting reaction product, and analyzing the purified product using a known technique such as liquid chromatography (LC).

The phrase “amino acid sequence wherein 1 to 77 amino acid residues have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 7 or 8” means that 1 to 77 amino acid residues have been deleted, substituted, inserted, and/or added at any 1 to 77 positions in the same sequence, wherein two or more of deletion, substitution, insertion, and addition may occur simultaneously.

Examples of amino acid residues that are interchangeable are as previously described.

The second active protein may not contain any secretory signal peptide or may contain a secretory signal peptide. When the protein of the present invention contains a secretory signal peptide, it preferably contains the secretory signal peptide at its N-terminus. The secretory signal peptide refers to a peptide domain that serves to cause extracellular secretion of a protein bound to the secretory signal peptide. Amino acid sequences of such secretory signal peptides and polynucleotide sequences encoding such amino acid sequences have been well known and reported in the art.

The second active protein can be obtained by, for example, expressing a polynucleotide encoding this protein (such as a polynucleotide consisting of SEQ ID NO: 5 or 6) in appropriate host cells, although it can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). The second active protein can also be chemically synthesized using a peptide synthesizer from AAPPTec LLC, Perkin Elmer Inc., Protein Technologies Inc., PerSeptive Biosystems, Applied Biosystems, or SHIMADZU CORPORATION, for example.

The “steviol glycoside” is as previously described. Examples of the steviol glycoside having a monoglucoside bond and/or monoglucosyl ester bond include rebaudioside A, stevioside, rubusoside, steviolmonoside, and steviol monoglucosyl ester.

The second active protein cleaves the monoglucoside bond and/or monoglucosyl ester bond. The resulting product varies depending on the starting material, the “steviol glycoside having at least one monoglucoside bond and/or monoglucosyl ester bond”, as follows. Examples thereof are shown below.

TABLE 3 Starting Material Product Rebaudioside A Rebaudioside B Stevioside Steviolbioside Rubusoside Steviol Steviolmonoside Steviol monoglucosyl ester

With the method for producing at least one of rebaudioside B and steviol comprising reacting the protein of the present invention, the second active protein, and one or more steviol glycosides selected from rebaudioside A, rebaudioside D, stevioside, rubusoside, steviolmonoside, and steviol monoglucosyl ester, based on the activity of the second active protein, rebaudioside B and steviol can be obtained as products from rebaudioside A, rebaudioside D, and stevioside as shown below.

TABLE 4 Starting Material Product Rebaudioside D Rebaudioside B Rebaudioside A Stevioside Steviol Rubusoside Steviolmonoside Steviol monoglucosyl ester

2. Method for Producing the Steviol Glycoside of the Present Invention Using a Non-Human Transformant

The protein of the present invention is a koji mold-derived secretory enzyme or a variant thereof, and is expected to have high activity in an extracellular environment. In this case, the steviol glycoside of the present invention can be produced by introducing a polynucleotide encoding the protein of the present invention (see “the polynucleotide of the present invention” described below) into host cells derived from bacteria, fungi, plants, insects, non-human mammals, or the like, for extracellular expression of the protein of the present invention, and by reacting the protein of the present invention with a steviol glycoside having a β-1,2-glucoside bond. Alternatively, depending on the host, the steviol glycoside of the present invention can be produced by expressing the protein of the present invention in the host cells.

Thus, the present invention provides a method for producing a steviol glycoside having no unbranched β-1,2-glucoside bond comprising culturing a non-human transformant (hereinafter referred to as “the transformant of the present invention”) obtained by introducing a polynucleotide selected from the group consisting of polynucleotides (a) to (e) shown below (hereinafter referred to as “the polynucleotide of the present invention”) into a host producing a steviol glycoside having at least one unbranched β-1,2-glucoside bond:

(a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;

(b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;

(c) a polynucleotide encoding a protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside;

(d) a polynucleotide encoding a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and

(e) a polynucleotide which hybridizes under highly stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and which encodes a protein having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside.

As used herein, the term “polynucleotide” refers to DNA or RNA.

Examples of the polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4 include a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1. Examples of the polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8 include a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5.

Examples of the “protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside” are as described above.

Examples of the “protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside” are as described above.

As used herein, the phrase “a polynucleotide which hybridizes under highly stringent conditions” refers to a polynucleotide obtained by means of a hybridization method such as colony hybridization, plaque hybridization, or Southern hybridization, using, as a probe, all of or a portion of a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 or 4. For hybridization, methods as described in “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 4, Cold Spring Harbor, Laboratory Press 2012” and “Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997”, for example, can be used.

As used herein, the term “highly stringent conditions” refers to, for example, the following conditions: 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, 50° C.; 0.2×SSC, 0.1% SDS, 60° C.; 0.2×SSC, 0.1% SDS, 62° C.; or 0.2×SSC, 0.1% SDS, 65° C.; although not limited thereto. Under these conditions, it is expected that DNA having a higher sequence identity will be efficiently obtained at a higher temperature. Note, however, that a plurality of factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration are considered to affect the stringency of hybridization, and a person skilled in the art will be able to achieve the same stringency by selecting these factors as appropriate.

When a commercially available kit is used for hybridization, the Alkphos Direct Labelling and Detection System (GE Healthcare), for example, can be used. In this case, hybridization is accomplished in accordance with the protocol attached to the kit, i.e., a membrane may be incubated overnight with a labeled probe and then washed with a primary washing buffer containing 0.1% (w/v) SDS at 55 to 60° C. to detect the hybridized DNA. Alternatively, when a commercially available reagent (e.g., PCR labeling mix (Roche Diagnostics)) is used for digoxigenin (DIG) labeling of a probe during probe preparation based on all of or a portion of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 of a nucleotide sequence complementary to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 or 4, the DIG nucleic acid detection kit (Roche Diagnostics) may be used for detection of hybridization.

In addition to those described above, examples of other hybridizable polynucleotides include DNA sharing 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity with DNA of the nucleotide sequence of SEQ ID NO: 1 or DNA encoding the amino acid sequence of SEQ ID NO: 3 or 4, as calculated by the homology search software BLAST using default parameters.

Note that the sequence identity of amino acid sequences or nucleotide sequences can be determined using the BLAST algorithm developed by Karlin and Altschul (Basic Local Alignment Search Tool) (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993). When BLAST is used, default parameters in each program are used.

The polynucleotide of the present invention may further contain a polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide. Preferably, the polynucleotide of the present invention contains, at its 5′ end, the polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide. The secretory signal peptide is as described above. Such a secretory signal peptide can be selected as appropriate, depending on the host into which the polynucleotide of the present invention is to be introduced. For example, when the host is yeast, examples of secretory signal peptides include yeast-derived secretory signal peptides, such as MF(ALPHA)1 signal peptide, PHO5 signal peptide, and SUC2 signal peptide. Examples of polynucleotides encoding MF(ALPHA)1 signal peptide, PHO5 signal peptide, and SUC2 signal peptide include polynucleotides consisting of the nucleotide sequences shown in SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 35, respectively. The amino acid sequences of MF(ALPHA)1 signal peptide, PHO5 signal peptide, and SUC2 signal peptide are shown in SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36, respectively. When the host is koji mold, examples of secretory signal peptides include koji mold-derived signal peptides, such as a peptide consisting of the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. The polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30 is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 29, respectively, for example.

The above-described polynucleotide of the present invention can be obtained using a known genetic engineering technique or a known synthesis technique.

The polynucleotide of the present invention is preferably inserted into an appropriate expression vector for introduction into a host.

An appropriate expression vector is typically configured to include:

(i) a promoter transcribable in host cells;

(ii) the polynucleotide of the present invention ligated to the promoter; and

(iii) an expression cassette containing, as constituent elements, signals that function in the host cells for transcription termination and polyadenylation of an RNA molecule.

Examples of methods for preparing such an expression vector include, although not particularly limited to, using plasmids, phages, cosmids, or the like.

The specific type of the vector is not particularly limited, and any vector expressible in host cells may be selected as appropriate. Specifically, an appropriate promoter sequence may be selected in accordance with the type of the host cells to ensure the expression of the polynucleotide of the present invention, and this promoter sequence and the polynucleotide of the present invention may then be integrated into any of various plasmids, for example, for use as an expression vector.

The expression vector of the present invention contains an expression control region (e.g., a promoter, a terminator, and/or a replication origin), depending on the type of the host into which the expression vector is to be introduced. For bacterial expression vectors, commonly used promoters (e.g., trc promoter, tac promoter, and lac promoter) are used. Examples of yeast promoters include glyceraldehyde-3-phosphate dehydrogenase promoter and PHO5 promoter. Examples of filamentous fungi promoters include amylase and trpC. Moreover, examples of promoters for expression of a target gene in plant cells include cauliflower mosaic virus 35S RNA promoter, rd29A gene promoter, rbcS promoter, and mac-1 promoter configured to have the enhancer sequence of the above-mentioned cauliflower mosaic virus 35S RNA promoter at the 5′-side of Agrobacterium-derived mannopine synthase promoter sequence. Examples of promoters for animal cell hosts include viral promoters (e.g., SV40 early promoter and SV40 late promoter). Examples of promoters inducibly activated by external stimulation include mouse mammary tumor virus (MMTV) promoter, tetracycline-responsive promoter, metallothionein promoter, and heat-shock protein promoter.

The expression vector preferably contains at least one selection marker. For use as such a marker, auxotrophic markers (ura5, niaD), drug resistance markers (hygromycin, zeocin), geneticin resistance gene (G418r), copper resistance gene (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA, vol. 81, p. 337, 1984), cerulenin resistance genes (fas2m, PDR4) (Junji Inokoshi et al., Biochemistry, vol. 64, p. 660, 1992; Hussain et al., Gene, vol. 101, p. 149, 1991), and the like are available.

While the method for preparing (producing) the transformant of the present invention is not particularly limited, the transformant of the present invention may be prepared by, for example, introducing an expression vector containing the polynucleotide of the present invention into a host to transform the host. The host to be used herein is not particularly limited as long as it produces a steviol glycoside having at least one unbranched β-1,2-glucoside bond, and may include not only a plant such as Stevia rebaudiana that produces a steviol glycoside having at least one unbranched β-1,2-glucoside bond, but also a host obtained by introducing a gene required for the production of a steviol glycoside having at least one unbranched β-1,2-glucoside bond into cells or an organism that does not originally produce a steviol glycoside having at least one unbranched β-1,2-glucoside bond. Examples of the “gene required for the production of a steviol glycoside having at least one unbranched β-1,2-glucoside bond” include genes having steviol or steviol glycoside synthesis activity such as those described in WO 2011/093509. Any of conventionally known various types of cells or organisms can be suitably used as the cells or organism to be transformed. Examples of the cells to be transformed include bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), filamentous fungi (koji mold Aspergillus oryzae, Aspergillus sojae), plant cells, and non-human animal cells. Appropriate media and conditions for culturing the above-described host cells are well known in the art. Likewise, the organism to be transformed is not particularly limited, and examples include various microorganisms, plants, and non-human animals described above as examples of host cells. The transformant is preferably yeast or a plant.

For transformation of the host cells, commonly used known methods can be used. For example, transformation can be accomplished using electroporation (Mackenxie, D. A. et al., Appl. Environ. Microbiol., vol. 66, p. 4655-4661, 2000), the particle delivery method (described in JP 2005-287403 A entitled “Breeding Method of Lipid Producing Fungi”), the spheroplast method (Proc. Natl. Acad. Sci. USA, vol. 75, p. 1929, 1978), the lithium acetate method (J. Bacteriology, vol. 153, p. 163, 1983), and other methods as described in Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual, although not limited thereto. When a gene is introduced into a plant or into tissues or cells derived from a plant, a method selected from the Agrobacterium method (Plant Molecular Biology Manual, Gelvin, S. B. et al., Academic Press Publishers), particle gun method, PEG method, electroporation, etc. can be used as appropriate.

For other standard molecular biological techniques, reference may be made to “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 4, Cold Spring Harbor Laboratory Press 2012” and “Methods in Yeast Genetics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)”, for example.

When the transformant is yeast or koji mold, the transformant is obtained by introducing a recombinant vector containing the polynucleotide of the present invention into yeast or koji mold such that a polypeptide encoded by the polynucleotide can be expressed. The yeast or koji mold transformed with the polynucleotide of the present invention expresses a higher level of the protein of the present invention than in the wild-type counterpart. Thus, the expressed protein of the present invention reacts with the steviol glycoside having at least one unbranched β-1,2-glucoside bond produced in the yeast or koji mold, thereby cleaving said unbranched β-1,2-glucoside bond. As a result, the steviol glycoside of the present invention having no unbranched β-1,2-glucoside bond is produced in the cells or culture medium of the yeast or koji mold, preferably in the culture medium.

When the transformant is a plant, the transformant is obtained by introducing a recombinant vector containing the polynucleotide of the present invention into a plant such that a protein encoded by the polynucleotide can be expressed. The plant to be transformed in the present invention refers to any of whole plants, plant organs (e.g., leaves, petals, stems, roots, and seeds), plant tissues (e.g., epidermis, phloem, parenchyma, xylem, vascular bundles, palisade tissue, and spongy parenchyma) or plant cultured cells, or various forms of plant cells (e.g., suspension cultured cells), protoplasts, leaf sections, calli, and the like. The plant used for transformation is not particularly limited as long as it is a plant that produces a steviol glycoside having at least one unbranched β-1,2-glucoside bond, or a plant that does not originally produce a steviol glycoside having at least one unbranched β-1,2-glucoside bond, but can produce a steviol glycoside having at least one unbranched β-1,2-glucoside bond through the introduction of a required gene. The plant used for transformation may be a plant in the class of either monocotyledons or dicotyledons. The introduction of the polynucleotide of the present invention into the plant can be confirmed by using PCR, Southern hybridization, or Northern hybridization, for example. Once a transformed plant in which the polynucleotide of the present invention has been integrated into the genome is obtained, progeny plants can be produced by sexual or asexual reproduction of the plant. Moreover, seeds, fruits, cuttings, tubers, root tubers, rootstocks, calli, protoplasts or the like can be obtained from this plant or progeny plants thereof, or clones thereof, and used to achieve mass production of the plant. The plant transformed with the polynucleotide of the present invention (hereinafter, “the plant of the present invention”) contains a greater amount of the protein of the present invention than in the wild-type counterpart. Thus, the protein of the present invention reacts with the steviol glycoside having at least one unbranched β-1,2-glucoside bond produced in the plant of the present invention, thereby cleaving said unbranched β-1,2-glucoside bond. As a result, the steviol glycoside of the present invention having no β-1,2-glucoside bond is produced in the plant.

The transformant in some embodiments of the present invention or the culture medium thereof has a content of the Steviol glycoside of the present invention higher than that in the wild-type counterpart, and an extract or the culture medium of the transformant contains a high concentration of the Steviol glycoside of the present invention. An extract of the transformant of the present invention can be obtained by homogenating the transformant with glass beads, a homogenizer, or a sonicator, for example, centrifuging the homogenate, and collecting the supernatant. When the Steviol glycoside of the present invention accumulates in the culture medium, the transformant and the culture supernatant may be separated using a standard method (e.g., centrifugation or filtration) after the completion of culture, thereby obtaining the culture supernatant containing the Steviol glycoside of the present invention.

The extract or culture supernatant thus obtained may be further subjected to a purification step. The Steviol glycoside of the present invention may be purified in accordance with a standard separation and purification method. Specific methods for purification are the same as described above.

3. Method for Preparing the Steviol Glycoside of the Present Invention Using an Enzyme Agent Derived from Non-Human Transformed Cells

The steviol glycoside of the present invention can be produced by using an enzyme agent derived from transformed cells expressing the protein of the present invention, which are obtained by introducing the polynucleotide of the present invention into host cells derived from bacteria, fungi, plants, insects, non-human mammals, or the like, for expression of the protein of the present invention, i.e., by contacting the enzyme agent derived from transformed cells expressing the protein of the present invention with a steviol glycoside having at least one unbranched β-1,2-glucoside bond. The “enzyme agent derived from transformed cells” is not limited as long as it is prepared using transformed cells, and contains the protein of the present invention. Examples of the enzyme agent include transformed cells themselves, a transformed cell homogenate itself, a transformed cell culture supernatant itself, and a purified product thereof. Thus, the present invention provides a method for producing a steviol glycoside comprising contacting an enzyme agent derived from a non-human transformed cell obtained by introducing, into a host cell, a polynucleotide selected from the group consisting of polynucleotides (a) to (e) shown below, with a steviol glycoside having at least one unbranched β-1,2-glucoside bond, thereby cleaving said unbranched β-1,2-glucoside bond:

(a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;

(b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;

(c) a polynucleotide encoding a protein consisting of an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside;

(d) a polynucleotide encoding a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and

(e) a polynucleotide which hybridizes under highly stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and which encodes a protein having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside.

The polynucleotide selected from the group consisting of polynucleotides (a) to (e) shown above is the polynucleotide of the present invention, which is the same as described above.

The polynucleotide of the present invention may further contain a polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide. Preferably, the polynucleotide of the present invention contains, at its 5′ end, the polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide. The secretory signal peptide and the polynucleotide consisting of a nucleotide sequence encoding the secretory signal peptide are the same as described above.

The polynucleotide of the present invention is preferably inserted into an appropriate expression vector for introduction into host cells. An appropriate expression vector is the same as described above.

While the method for preparing the transformed cells of the present invention is not particularly limited, the transformed cells of the present invention may be prepared by, for example, introducing an expression vector containing the polynucleotide of the present invention into host cells to transform the host cells. The cells to be transformed are the same as described above. The method for transforming the host cells is as described above.

The transformed cells of the present invention are obtained by, for example, introducing a recombinant vector containing the polynucleotide of the present invention into the host cells such that a polypeptide encoded by the polynucleotide can be expressed. The host cells transformed with the polynucleotide of the present invention express a higher level of the protein of the present invention than in the wild-type counterpart. Thus, the steviol glycoside of the present invention can be obtained by using an enzyme agent derived from transformed cells expressing the protein of the present invention, i.e., by contacting the enzyme agent derived from transformed cells expressing the protein of the present invention with a steviol glycoside having at least one unbranched β-1,2-glucoside bond.

The term “contact” refers to causing the enzyme agent derived from the transformed cells of the present invention and the steviol glycoside having at least one unbranched β-1,2-glucoside bond to exist in the same reaction or culture system. The term “contact” includes, for example, adding the steviol glycoside having at least one unbranched β-1,2-glucoside bond to a container containing the enzyme agent derived from the transformed cells of the present invention, mixing the enzyme agent derived from the transformed cells of the present invention and the steviol glycoside having at least one unbranched β-1,2-glucoside bond, and adding the enzyme agent derived from the transformed cells of the present invention to a container containing the steviol glycoside having at least one unbranched β-1,2-glucoside bond.

The terms “steviol glycoside” and “steviol glycoside having at least one unbranched β-1,2-glucoside bond” are the same as described above.

The Steviol glycoside of the present invention thus obtained can be used for such purposes as the production of foods, sweeteners, flavors, pharmaceutical products, and industrial raw materials (raw materials for cosmetics, soaps, and the like), for example, in accordance with conventional methods.

Examples of foods include nutritional supplements, health foods, functional foods, foods for children, and foods for the elderly. As used herein, the term “foods” refers collectively to edible materials in the form of solids, fluids, liquids, and mixtures thereof.

Note that all documents, as well as laid-open application publications, patent application publications, and other patent documents cited herein shall be incorporated herein by reference.

Examples

The present invention will be more specifically described hereinafter with reference to examples, which are not intended to limit the scope of the present invention.

1. Cloning of Steviol-Hydrolyzing Enzyme Genes

1-(1) Genes

AO090009000356 and AO090701000274 were cloned and expressed in yeast to examine the activity.

1-(2) Cloning of cDNAs of Koji Mold

Koji mold Aspergillus oryzae var. Brunneus (IFO30102) was inoculated to a GY plate (2% glucose, 0.5% yeast extract, and 2% agar), and cultured at 25° C. for 3 days. The grown cells were collected from the GY plate, and total RNA was extracted using RNeasy (QIAGEN). A cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies).

The following primers were designed based on the DNA sequence of AO090009000356 and AO090701000274:

AO090009000356 (hereinafter expressed as AOBGL1) AOBGL1-1: (SEQ ID NO: 9) 5′-AGATCTATGAAGCTTGGTTGGATCGAGGT-3′ AOBGL1-2: (SEQ ID NO: 10) 5′-GTCGACTTACTGGGCCTTAGGCAGCGA-3′ AO090701000274 (hereinafter expressed as AOBGL3) AOBGL3-1: (SEQ ID NO: 11) 5′-GCGGCCGCatggtttccggtgtctttacgaagg-3′ AOBGL3-2: (SEQ ID NO: 12) 5′-GGATCCtcactgcacatagaaagtagcattgcc-3′

Approximately 2.6 kbp or 2.3 kbp of a DNA fragment amplified by PCR using ExTaq (Takara Bio), using each of the cDNAs synthesized as described above as a template, was cloned using the TOPO-TA cloning Kit (Life Technologies). Each of the plasmids obtained herein was designated as pCR-AOBGL1 or pCR-AOBGL3.

2. Expression in Yeast

2-(1) Construction of Yeast Expression Vectors and Acquisition of Transformed Strains

A DNA fragment obtained by digesting the yeast expression vector pYE22m (Biosci. Biotech. Biochem., 59, 1221-1228, 1995) with restriction enzymes BamHI and SalI and approximately 2.6 kbp of a DNA fragment obtained by digesting pCR-AOBGL1 with restriction enzymes BglII and SalI were ligated using the DNA Ligation Kit Ver.1 (Takara Bio), and the resulting plasmid was designated as pYE-AOBGL1. Additionally, a DNA fragment obtained by digesting pYE22 mN with restriction enzymes NotI and BamHI and approximately 2.3 kbp of a DNA fragment obtained by digesting pCR-AOBGL3 with restriction enzymes NotI and BamHI were ligated in the same manner as above, and the resulting plasmid was designated as pYE-AOBGL3. S. cerevisiae strain EH13-15 (trp1, MATα) (Appl. Microbiol. Biotechnol., 30, 515-520, 1989) was used as the parental strain for transformation.

Each of the plasmids pYE22m (control), pYE-AOBGL1 (for expression of AOBGL1), and pYE-AOBGL3 (for expression of AOBGL3) was used to transform strain EH13-15 in accordance with the lithium acetate method. A strain that grew on SC-Trp (containing, per liter, 6.7 g of Yeast nitrogen base w/o amino acids (DIFCO), 20 g of glucose, and 1.3 g of amino acid powder (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 1.8 g of leucine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, and 0.6 g of uracil) agar medium (2% agar) was selected as the transformed strain.

The selected strain was applied to SC-Trp agar medium containing 0.004% of X-β-Glc, and cultured at 30° C. for 3 days. As a result, neither the strain transformed with any of the plasmids pYE-AOBGL1 and pYE-AOBGL3 nor the strain transformed with the control pYE22m was stained blue, and no X-β-Glc degrading activity was confirmed.

Meanwhile, one platinum loop of the selected strain was inoculated to 10 mL of SC-Trp liquid medium supplemented with 1/10 volume of 1 M potassium phosphate buffer, and cultured with shaking at 30° C. and 125 rpm for 2 days. The resulting culture was separated into the culture supernatant and cells by centrifugation. The cells were suspended in 50 mM sodium phosphate buffer (pH 7.0) containing 0.1% CHAPS solution and then homogenated with glass beads, and the supernatant obtained by centrifugation was used as the cell homogenate. The obtained culture supernatant or cell homogenate was examined for its pNP-β-Glc activity.

As a result, both the culture supernatant and cell homogenate for each type of transformed strain including the control exhibited pNP-β-Glc degrading activity, and no significant difference in activity was observed between them.

These results suggested that the introduction of the plasmid pYE-AOBGL1 or pYE-AOBGL3 into yeast strain EH13-15 does not allow expression of an activated protein having β-glucosidase activity. Moreover, the need for the deletion of an endogenous gene responsible for β-glucosidase activity in yeast was indicated.

2-(2) Creation of ΔExg1 ΔExg2 Yeast Host Strains

A strain with deletion of the EXG1 (YLR300w) gene considered to be responsible for most of the extracellular β-glucosidase activity in yeast and its homolog EXG2 (YDR261c) gene was used as the host strain for transformation. This host strain was created as follows:

Each of Δexg1 strain (MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0; clone ID: 15210; Open Bio Systems) and Δexg2 strain (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0; clone ID: 3620; Open BioSystems) was applied to YPD agar medium, and cultured at 30° C. for 2 days. The cells of each strain were scraped with a platinum loop and mixed on SC-Met, Lys (containing, per liter, 6.7 g of Yeast nitrogen base w/o amino acids (DIFCO), 20 g of glucose, and 1.3 g of amino acid powder (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 1.8 g of leucine, 1.5 g of phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, 1.2 g of tryptophan, and 0.6 g of uracil) agar medium (2% agar), and the mixture was cultured at 30° C. for 2 days. The grown strain was considered to be a hetero-diploid obtained by hybridization of the two strains. The obtained strain was applied to YPD agar medium and cultured at 30° C. for 2 days, and then the cells were scraped with a platinum loop, applied to 0.5% potassium acetate agar medium (2% agar), and cultured at room temperature for 5 days, thus forming spores. Tetrad dissection was performed to separate haploid strains. Genotypes of the obtained strains were confirmed by PCR, and Δexg1 Δexg2-1 strain (his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) was selected.

Using the genomic DNA of yeast strain S288C, PCR was performed with the following primers TRP1-F and TRP1-R, using KOD-Plus (Toyobo). Approximately 2.7 kbp of the amplified DNA fragment was cloned using the Zero Blunt TOPO PCR cloning Kit (Life Technologies), thus obtaining a plasmid pCR-TRP1.

TRP1-F: (SEQ ID NO: 13) TACTATTAGCTGAATTGCCACTGCTATCG TRP1-R: (SEQ ID NO: 14) TCTACAACCGCTAAATGTTTTTGTTCG

2.7 kbp of a DNA fragment obtained by digesting pPRGINFRT3-103 (Japanese Patent Laid-Open No. 2001-120276) with restriction enzymes EcoRI and HindIII was blunt-ended using the Blunting Kit (Takara Bio), and then ligated to a DNA fragment obtained by digesting the plasmid pCR-TRP1 with restriction enzymes HpaI and StuI, using Ligation High (Toyobo), thus obtaining a plasmid pCR-Δtrp1:URA3-FRT. Using this plasmid as a template, PCR was performed with the primers TRP1-F and TRP1-R, using KOD-Plus (Toyobo). Then, 4.4 kbp of the resulting DNA fragment was used to transform Δexg1 Δexg2-1 strain in accordance with the lithium acetate method, and a strain that grew on SC-Ura (containing, per liter, 6.7 g of Yeast nitrogen base w/o amino acids (DIFCO), 20 g of glucose, and 1.3 g of amino acid powder (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 1.8 g of leucine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, and 1.2 g of tryptophan) agar medium (2% agar) was selected as the transformed strain. The transformed strain was cultured on YPGal medium (yeast extract: 2%, polypeptone: 1%, galactose: 2%) and then applied to SC+5-FOA (containing, per liter, 6.7 g of Yeast nitrogen base w/o amino acids (DIFCO), 20 g of glucose, and 1.3 g of amino acid powder (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 1.8 g of leucine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, 1.2 g of tryptophan, and 0.6 g of uracil) agar medium (2% agar), and a grown strain was obtained as Δexg1 Δexg2-2 strain and used as the host for the following transformation.

2-(3) Substitution of Secretory Signal Sequences and Expression in Yeast

Nineteen amino acids (SEQ ID NO: 28) at the N-terminus of AOBGL1p and 22 amino acids (SEQ ID NO: 30) at the N-terminus of AOBGL3 are estimated to be secretory signal sequences.

Thus, for secretion and expression of each of AOBGL1 and AOBGL3 in yeast, the estimated secretory signal sequence was substituted with a secretory signal sequence of a yeast secretory protein. Initially, the following oligodeoxynucleotides were synthesized and annealed, and then inserted into the EcoRI site of the vector pYE22m, thus creating pYE-PacNhe.

PacI-NheI-F: (SEQ ID NO: 15) 5′-AATTAATTAAGAGCTAGCG-3′ PacI-NheI-R: (SEQ ID NO: 16) 5′-TTAATTCTCGATCGCTTAA-3

Using the plasmid pCR-AOBGL1 or pCR-ASBGL1 as a template, PCR was performed with the following primers Bgl2-AOBGL1-F and AOBGL1-2, using KOD-Plus (Toyobo). Approximately 2.5 kbp of a DNA fragment obtained by digesting the PCR-amplified DNA fragment with restriction enzymes BglII and SalI was inserted into the sites of restriction enzymes BamHI and SalI of the vector pYE-PacNhe, thus constructing a plasmid pYE-PN-AOBGL1.

Bgl2-AOBGL1-F: (SEQ ID NO: 17) 5′-TAAGATCTAAGGATGATCTCGCGTACTCCCC-3 AOBGL1-2: (SEQ ID NO: 18) 5′-GTCCACTTACTGGGCCTTAGGCAGCGA-3′

Using the plasmid pCR-ASBGL3 as a template, PCR was performed with the following primers Bam-ASBGL3-F and Sal-ASBGL3-R, using KOD-Plus (Toyobo). Approximately 2.3 kbp of a DNA fragment obtained by digesting the PCR-amplified DNA fragment with restriction enzymes BamHI and SalI was inserted into the sites of restriction enzymes BamHI and SalI of the vector pYE-PacNhe, thus constructing a plasmid pYE-PN-AOBGL3.

Bam-AOBGL3-F: (SEQ ID NO: 19) 5′-AAGGATCCCAAGATGAGAAGCCTCGCTACAAGG-3 Sal-AOBGL3-R: (SEQ ID NO: 20) 5′-GGGTCGACTCACTGCACATAGAAAGTAGCATTGCC-3′

The primers shown below were designed to construct a plasmid for expression of a protein in which the estimated secretory signal sequence of AOBGL1 or AOBGL3 was substituted with the secretory signal sequence MF(ALPHA)1 (YPL187W) (the sequence of positions 1 to 19 of the amino acid sequence (SEQ ID NO: 32) shown in FIG. 2A), PHO5 (YBR093C) (the sequence of positions 1 to 17 of the amino acid sequence (SEQ ID NO: 34) shown in FIG. 2B), or SUC2 (YIL162W) (the sequence of positions 1 to 19 of the amino acid sequence (SEQ ID NO: 36) shown in FIG. 2C) of a yeast secretory protein.

ScPHO5-F: (SEQ ID NO: 21) 5′- TAAATGTTTAAATCTGTTCTTTATTCAATTTTAGCCGCTTCTTTCCCCAA TGCAG-3′ ScPHO5-R: (SEQ ID NO: 22) 5′- CTAGCTGCATTGGCCAAAGAACCCGCTAALATTGAATAAACAACAGATTT AAACATTTAT-3′ ScSUC2-F: (SEQ ID NO: 23) 5′- TAAATGCTTTTGCAAGCTTTCCTTTTCCTTTTGGCTGGTTTTGCACCCAA AATATCTCCAG-3′ ScSUC2-R: (SEQ ID NO: 24) 5′- TAAATGAGATTTCCTTCAATTTTTACTCCACTTTTATTCGCAGCATCCTC CGCATTAGCTG-3′ ScMF1-F: (SEQ ID NO: 25) 5′- TAAATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTC CGCATTAGCTG-3′ ScMF1-R: (SEQ ID NO: 26) 5′- CTAGCAGCTAATCCCGAGCATGCTCCGAATAAAACTGCAGTAAAAATTGA AGGAAATCTCATTTAAT-3′

The combination of ScPHO5-F and ScPHO5-R, the combination of ScSUC2-F and ScSUC2-R, and the combination of ScMF1-F and ScMF1-R were each annealed, and then ligated to the plasmid pYE-PN-AOBGL1 or pYE-PN-ASBGL1 digested with restriction enzymes PacI and NheI, thus obtaining the following plasmids:

pYE-PHO5s-AOBGL1 (for expression of PHO5s-AOBGL1)

pYE-SUC2s-AOBGL1 (for expression of SUC2s-AOBGL1)

pYE-MF1s-AOBGL1 (for expression of MF1s-AOBGL1)

pYE-PHO5s-AOBGL3 (for expression of PHO5s-AOBGL3)

pYE-SUC2s-AOBGL3 (for expression of SUC2s-AOBGL3)

pYE-MF1s-AOBGL3 (for expression of MF1s-AOBGL3)

Δexg1 Δexg2-2 strain was transformed with these plasmids in accordance with the lithium acetate method, and a strain that grew on SC-Trp agar medium was selected as the transformed strain.

The obtained transformed strain was applied to SD-Trp agar medium containing 0.004% of X-β-Glc, and cultured at 30° C. for 3 days. As a result, the cells and surrounding regions were stained blue in the strain transformed with pYE-PHO5s-AOBGL1, pYE-SUC2s-AOBGL1, pYE-MF1s-AOBGL1, pYE-PHO5s-ASBGL1, pYE-SUC2s-ASBGL1, or pYE-MF1s-ASBGL1, suggesting that these strains had X-β-Glc hydrolyzing activity.

Meanwhile, in the strain transformed with the plasmid for expression of AOBGL3, the cells and surrounding regions were not stained blue, and the activity to hydrolyze X-β-Glc was not confirmed.

In the strain transfected with a control vector, the cells and surrounding regions were not stained blue, showing that the strain did not have the activity to hydrolyze X-β-Glc.

One platinum loop of the obtained transformed strain was inoculated to a liquid medium obtained by mixing 10 mL of SD-Trp liquid medium and 1 mL of 1 M potassium phosphate buffer, and cultured with shaking at 30° C. for 2 days. The culture was separated into the cells and culture supernatant by centrifugation. 50 μL of the culture supernatant, 50 μL of a 0.2 M sodium citrate buffer, 50 μL of a 20 mM aqueous pNP-βGlc solution, and 50 μL of water were mixed, and the mixture was reacted at 37° C. for 1 hour, after which the increase in absorbance at 405 nm due to β-glucosidase activity was examined. When AOBGL1 was expressed, the β-glucosidase activity was confirmed as shown in Table 5. AOBGL1 was considered to be expressed at the highest level when substituted with the MF1 signal sequence.

TABLE 5 β-glucosidase activity in culture medium (Increase in absorbance at 405 nm; reaction for 1 hour) Plasmid Δ 405 nm pYE-MF1s-AOBGL1 0.508 pYE-PHO5s-AOBGL1 0.37 pYE-SUC2s-AOBGL1 0.369 pYE22m 0

However, when AOBGL3 was expressed, the β-glucosidase activity was not confirmed under the same conditions.

2-(4) Activity of Recombinant Enzyme on Steviol Glycoside

AOBGL1 was confirmed to have an activity to convert stevioside into rubusoside.

3. Expression of AOBGL3 in Koji Mold

3-(1) Construction of Koji Mold Expression Vector

A DNA fragment obtained by digesting a koji mold vector pUNA (National Research Institute of Brewing) with a restriction enzyme SmaI, and approximately 2.3 kbp of a DNA fragment obtained by digesting the plasmid pCR-AOBGL3 with restriction enzymes NotI and BamHI and blunt-ending the end using Blunting Kit (Takara Bio), were ligated to obtain a plasmid pUNA-AOBGL3.

3-(2) Transformation of Koji Mold

Koji mold was transformed as follows.

Aspergillus oryzae niaD300 strain (National Research Institute of Brewing) was used as a host. The host strain was inoculated to a PDA plate and cultured at 30° C. for about 1 week. In order to obtain a conidial suspension, the resulting conidia were suspended by adding 0.1% tween 80 and 0.8% NaCl. The suspension was filtered through Miracloth and then centrifuged to collect the conidia. The conidia were then washed with 0.1% tween 80 and 0.8% NaCl and suspended in sterilized water.

The conidia were applied to a CD plate, and DNA was introduced into the conidia by the particle delivery method. This was performed using PDS-1000/He (Bio-Rad), tungsten M-10 particles, and a 1100 psi rupture disc at a distance of 3 cm. A strain that grew on a CD plate (containing, per liter, 6 g of NaNO₃, 0.52 g of KCl, 1.52 g of KH₂PO₄, 10 g of glucose, 2 ml of IM MgSO₄, 1 ml of a trace element solution (containing, per liter, 1 g of FeSO₄.7H₂O, 8.8 g of ZnSO₄.7H₂O, 0.4 g of CuSO₄.5H₂O, 0.1 g of NaB₄O₇.10H₂O, and 0.05 g of (NH₄)₆Mo₇O₂₄.4H₂O), and 20 g of agar (pH 6.5)) was selected as the transformed strain.

3-(3) Preparation of Conidial Suspension

BGL3-1 stain or C-1 strain was inoculated to a CD plate and cultured at 30° C. for 7 days to form conidia. In order to obtain a conidial suspension, the conidia were suspended by adding 0.1% tween 80 and 0.8% NaCl. The suspension was filtered through Miracloth and then centrifuged to collect the conidia. The conidia were then washed with 0.1% tween 80 and 0.8% NaCl and suspended in sterilized water to prepare a conidial suspension.

3-(4) Production of AOBGL3 by Liquid Culture

Conidia of BGL3-1 strain or C-1 strain were inoculated to a liquid culture medium for enzyme production (containing, per liter, 100 g of maltose, 1 g of Bacto-tryptone, 5 g of yeast extract, 1 g of NaNO₃, 0.5 g of K₂HPO₄, 0.5 g of MgSO₄.7H₂O, and 0.01 g of FeSO₄.7H₂O) and cultured with shaking at 30° C. for 2 days. The medium was filtered through Miracloth to remove the cells and further filtered through a membrane filtration system (IWAKI) to collect the supernatant. The supernatant was then concentrated by ultrafiltration through Amicon Ultra-15 50 k (Merck), and the buffer was replaced with a 50 mM sodium phosphate buffer (pH 7.0) containing 0.1% CHAPS to obtain a crude enzyme solution.

3-(5) Examination of Activity (Liquid Culture)

The activity on various substrates was examined. Both the pNP-β-Glc:BGL3-1 strain and C-1 strain were stained yellow, showing that both had the activity to hydrolyze pNP-β-Glc.

The X-β-Glc:BGL3-1 strain was stained blue, showing that it had the activity to hydrolyze X-β-Glc. By contrast, the C-1 strain was not stained blue, which was considered to indicate that the C-1 strain did not have the activity to hydrolyze X-β-Glc. The activity on X-β-Glc was attributed to an AOBGL3 gene product.

3-(6) Activity to Hydrolyze Steviol Glycoside

An enzyme solution derived from BGL3-1 strain or C-1 strain (liquid culture or plate culture) was examined for the activity to hydrolyze steviol glycosides (rubusoside) as follows.

Reaction Conditions

50 μg/ml of substrate, 20 μl of the enzyme solution, and a 50 mM sodium citrate buffer (pH 5.0) were mixed to a total volume of 100 μl, and the mixture was reacted at 50° C. The reaction mixture was passed through Sep-Pak C18 (Waters) washed with acetonitrile and equilibrated with water. The reaction product was subsequently washed with 20% acetonitrile and then eluted with 50% acetonitrile. The eluate was evaporated to dryness with SpeedVac. The resulting product was dissolved in 100 μL of 80% acetonitrile, and the solution was subjected to HPLC.

The conditions for HPLC were as follows:

Column: COSMOSIL 5C₁₈-AR-II 4.6 mm I.D.×250 mm (Nacalai Tesque)

Mobile phase: A; acetonitrile, B;

B conc. 70%→30% 40 min linear gradient

Flow rate: 1 ml/min

Temperature: 40° C.

Detection: UV 210 nm

In summary, the enzyme activity to hydrolyze the glucoside bond at the 13 position of rubusoside and the glucosyl ester bond at the 19 position of rubusoside was exhibited through expression of AOBGL3. In view of the fact that an intermediate, steviol monoglucosyl ester, was detected in addition to steviol in the reaction with rubusoside, the AOBGL3 protein was found to be an enzyme which, although capable of hydrolyzing both the glucoside bond at the 13 position of rubusoside and the glucosyl ester bond at the 19 position of rubusoside, preferentially hydrolyzes the glucoside bond at the 13 position. 

The invention claimed is:
 1. A method for producing a steviol glycoside having no unbranched β-1,2-glucoside bond, the method comprising: reacting a protein selected from the group consisting of proteins (a) to (c) shown below with a steviol glycoside having at least one unbranched β-1,2-glucoside bond, thereby cleaving said unbranched β-1,2-glucoside bond: (a) a protein comprising the amino acid sequence of SEQ ID NO: 3 or 4; (b) a protein comprising an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and (c) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside.
 2. The method of claim 1, wherein the protein selected from the group consisting of the proteins (a) to (c) further comprises a secretory signal peptide.
 3. The method of claim 2, wherein the secretory signal peptide is a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 28, 30, 32, 34, and
 36. 4. The method of claim 3, wherein the steviol glycoside having at least one unbranched β-1,2-glucoside bond is selected from rebaudioside D, rebaudioside E, stevioside, and steviolbioside.
 5. The method of claim 4, wherein the steviol glycoside having no unbranched β-1,2-glucoside bond is selected from rubusoside, rebaudioside A, and steviolmonoside.
 6. A method for producing at least one of rebaudioside B and steviol comprising reacting together: a protein selected from the group consisting of proteins (a) to (c) shown below, a protein selected from the group consisting of proteins (d) to (f) shown below, and a steviol glycoside having at least one unbranched β-1,2-glucoside bond: (a) a protein comprising the amino acid sequence of SEQ ID NO: 3 or 4; (b) a protein comprising an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; (c) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; (d) a protein comprising the amino acid sequence of SEQ ID NO: 7 or 8; (e) a protein comprising an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 7 or 8, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside; and (f) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 7 or 8, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside.
 7. The method of claim 6, wherein the steviol glycoside having at least one unbranched β-1,2-glucoside bond comprises one or more steviol glycosides selected from rebaudioside D, rebaudioside E, stevioside, and steviolbioside. 